Pharmaceutical preparation comprising recombinant hcg

ABSTRACT

The present disclosure describes recombinant human chorionic gonadotropin (hCG) and methods for the production thereof. The recombinant hCG can include α2,3, α2,6, and, optionally, α2,8 sialylation. The recombinant hCG can be produced in a human cell line such as a PER.C6® cell line.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/283,904 filed on May 21, 2014, which is a continuation of U.S.application Ser. No. 13/500,274, now U.S. Pat. No. 8,975,226, filed onApr. 4, 2012 under 35 U.S.C. § 371 as the national phase of PCTInternational Application No. PCT/GB2010/001854, filed on Oct. 4, 2010,which claims the benefit of European Patent Application No. 09252360.4,filed on Oct. 5, 2009. The prior applications are incorporated herein byreference in their entirety.

FIELD

The present invention relates to gonadotrophins for use in the treatmentof infertility. In particular, it relates to human chorionicgonadotrophin (hCG).

BACKGROUND

The gonadotrophins are a group of heterodimeric glycoprotein hormoneswhich regulate gonadal function in the male and female. They includefollicle stimulating hormone (FSH), luteinising hormone (LH) andchorionic gonadotrophin (CG).

Human chorionic gonadotrophin (hCG) is naturally secreted by theanterior pituitary gland and functions to support follicular developmentand ovulation. hCG comprises a 92 amino acid alpha sub-unit, also commonto the other glycoprotein hormones LH and FSH, and a 145 amino acid betasub-unit unique to hCG, which dictates the hormone specificity. Eachsub-unit is post translationally modified by the addition of complexcarbohydrate residues. The alpha sub-unit contains 2-N-linkedglycosylation sites at amino acids 52 and 78 and the beta sub-unitcontains 2-N-linked glycosylation sites at amino acids 13 and 30 andfour O-linked glycosylation sites at amino acids 121, 127, 132 and 138.

hCG extracted from the urine of pregnant women [CHORAGON® (Ferring)] hasbeen used for many years in infertility treatment. The production of hCGextracted from urine involves the collection and processing of largeamounts of urine. A recombinant version of hCG, OVITRELLE® (Serono), isavailable. This is expressed in Chinese hamster ovary (CHO) cells. Theknown recombinant hCG product has a different pharmacokinetic profile tohCG produced from humane urine. It is desirable to have an hCG productthat more closely replicates or mimics the pharmacokinetic profile ofthe product produced from human urine.

There is considerable heterogeneity associated with hCG preparationswhich relates to differences in the amounts of various isoforms present.Individual hCG isoforms exhibit identical amino acid sequences butdiffer in the extent to which they are post-translationally modified;particular isoforms are characterised by heterogeneity of thecarbohydrate branch structures and differing amounts of sialic acid (aterminal sugar) incorporation, both of which appear to influence thespecific isoform bioactivity.

Glycosylation of natural hCG is highly complex. The glycans in naturallyderived pituitary hCG can contain a wide range of structures that caninclude combinations of bi-, tri- and tetra-antennary glycans. Theglycans can carry further modifications: core fucosylation, bisectingglucosamine, chains extended with acetyl lactosamine, partial orcomplete sialylation, sialylation with α2,3 and α2,6 linkages, andsulphated galactosamine substituted for galactose. Furthermore, thereare differences between the distributions of glycan structures at theindividual glycosylation sites.

The glycosylation of recombinant hCG (“rhCG”) products reflects therange of glycosyl-transferases present in the host cell line. Theexisting rhCG product, OVITRELLE®, is derived from engineered Chinesehamster ovary cells (CHO cells). The range of glycan modifications inCHO derived rhCG are more limited than those found on the naturalproducts, derived from urine. Examples of the reduced glycanheterogeneity found in CHO derived rhCG include a lack of bisectingglucosamine and a reduced content of core fucosylation and acetyllactosarnine extensions. In addition, CHO cells are only able to addsialic acid using the α2,3 linkage (Kagawa et al., 1988, Takeuchi etal., 1988, Svensson et al., 1990). This is different from naturallyproduced hCG which contains glycans with a mixture of α2,3 andα2,6-linked sialic acid.

It has been demonstrated that a recombinant FSH preparation (Organon)differs in the amounts of FSH with an isoelectric point (pI) of below 4(considered the acidic isoforms) when compared to pituitary, serum orpost-menopausal urine FSH (Ulloa-Aguirre et al., 1995). The amount ofacidic isoforms in the urinary preparations of FSH was much higher ascompared to the recombinant products, GONAL-F® (Serono) and PUREGON®(Organon) (Andersen et al., 2004). This must reflect a lower molarcontent of sialic acid in rFSH since the content of negatively-chargedglycan modified with sulphate is low in FSH. The lower sialic acidcontent, compared to natural FSH, is a feature of both commerciallyavailable FSH products and therefore must reflect a limitation in themanufacturing process (Bassett and Driebergen, 2005). The circulatorylife-time of FSH has been documented for materials from a variety ofsources. Some of these materials have been fractionated on the basis ofoverall molecular charge, as characterised by their pI, in which moreacid equates to a higher negative charge. The major contributor tooverall molecular charge is the total sialic content of each FSHmolecule. For instance, rFSH (Organon) has a sialic acid content ofaround 8 mol/mol, whereas urine-derived FSH has a higher sialic acidcontent (de Leeuw et al. 1996). The corresponding plasma clearance ratesin the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al., 2003). Inanother example where a sample of recombinant FSH was split into highand low pI fractions, the in vivo potency of the high pI (lower sialicacid content) fraction was decreased and it had a shorter plasmahalf-life (D'Antonio et al., 1999). The applicants have found that,similar to FSH, the known, CHO derived, recombinant hCG product (e.g.,OVITRELLE®) also has a lower amount of hCG with an isoelectric point(pI) of below 4 (considered the acidic isoforms) than urinary hCG, alsoreflecting a lower sialic acid content of the known rhCG productcompared to urinary hCG.

The total sialic acid content of hCG and rhCG is not directly comparablesince sialic acids are commonly linked in two ways.Pituitary/serum/urinary hCG contain both α2,3 and α2,6-linked sialicacid, with a predominance of the former. However, CHO cell derivedrecombinants only contain α2,3 (Kagawa et al., 1988, Takeuchi et al.,1988, Svensson et al., 1990). In other words, recombinant proteinsexpressed using the CHO system will differ from their naturalcounterparts in their type of terminal sialic acid linkages. This isanother difference between natural and current recombinant products inaddition to the lower overall sialic acid content of the latter, and isan important consideration in the production of biologicals forpharmaceutical use since the carbohydrate moieties may contribute to thepharmacological attributes of the molecule.

SUMMARY

It is therefore desirable to have a rhCG product that more closelyreplicates or mimics the physiochemical and pharmacokinetic profile ofthe product produced from human urine. It is desirable to have a rhCGproduct that has improved pharmacokinetic property or propertiescompared to the known recombinant product.

According to the present invention there is provided recombinant hCG(“rhCG” or “rechCG”) including α2,3 sialylation and α2,6 sialylationand, optionally. α2,8 sialylation. The rhCG (or rhCG preparation)according to the invention may have a sialic acid content [expressed interms of a ratio of moles of sialic acid to moles of protein] of 15mol/mol or greater, for example of from 15 mol/mol to 25 mol/mol, forexample from 17 mol/mol to 24 mol/mol, for example from 17.7 mol/mol to23 mol/mol, for example from 18 mol/mol to 22 mol/mol, for example from19 mol/mol to 21 mol/mol, for example from 19 mol/mol to 20 mol/mol. TherhCG (or rhCG preparation) according to the invention may have 10% ormore of the total sialylation being α2,3-sialylation. For example, 45%to 80% of the total sialylation may be α2,3-sialylation, for example 50%to 70% of the total sialylation may be α2,3-sialylation, for example 55to 65% of the total sialylation may be α2,3-sialylation. For example65-85% of the total sialylation may be α2,3-sialylation. The rhCG (orrhCG preparation) of the invention may have 50% or less of the totalsialylation being α2,6-sialylation. For example, 20-55% of the totalsialylation may be α2,6-sialylation, for example, 30-50% of the totalsialylation may be α2,6-sialylation, for example, 35-45% of the totalsialylation may be α2,6-sialylation. For example 15-35% of the totalsialylation may be α2,6-sialylation. The rhCG (or rhCG preparation) ofthe invention may have 5% or less of the total sialylation beingα2,8-sialylation, for example 0 to 4%, e.g.; 0.1-4% of the totalsialylation may be α2,8-sialylation. The rhCG (or rhCG preparation) ofthe invention may have no α2,8-sialylation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plasmid map of the phCGalpha/beta expression vector.

FIG. 2 is a representation of the α2,3-sialyltransferase (ST3GAL4)expression vector.

FIG. 3 is a representation of the α2,6-sialyltransferase (ST6GAL1)expression vector.

FIG. 4 is a representation of an isoelectric focusing gel, withCoomassie Blue staining, comparing rhCG Isoforms in human cell linederived recombinant hCG preparations according to the invention (track3, 4) with preparations of the prior art (track 1, 2).

FIG. 5 is a graph that shows metabolic clearance rates (MCRs) ofα2,3-sialytransferase engineered PER.C6® cell produced hCG samples; and

FIG. 6 is a graph that shows long term MCRs of α2,3 sialyltransferaseengineered PER.C6 cell prepared rhCG samples.

DESCRIPTION

The applicants have developed a human derived recombinant hCG which hasa more acidic profile than the CHO derived product, OVITRELLE®, andwhich has a higher sialic acid content. The applicants' researchindicates that the type of sialic acid linkage, α2,3- or α2,6-, can havea dramatic influence on biological clearance of hCG. Human cell lines,as opposed to CHO cell lines, can express recombinant hCG with sialicacids attached by both α2,3 and α2,6 linkages.

Recombinant hCG with a mixture of both α2,3 and α2,6-linked sialic acidwas made by engineering a human cell line to express both rhCG and α2,3sialyltransferase (Examples 4, 5a and 5b). The expressed product ishighly acidic and carries a mix of both α2,3- and α2,6-linked sialicacids; the latter provided by the endogenous sialyl transferaseactivity. This has two advantages over rhCG expressed in conventionalCHO cells: first the material is more highly sialylated due to thecombined activities of the two sialyltransferases; and secondly thematerial more closely resembles the natural hCG. This is likely to bemore biologically appropriate compared to CHO cell derived recombinantproducts that have produce only α2,3 linked sialic acid and havedecreased sialic acid content.

The applicants have surprisingly found that rhCG of the invention maymore closely replicate or mimic the physiochemical and pharmacokineticprofile of the natural human urinary product than other recombinantproducts. In other words, rhCG of the invention may be closer to the“natural” hCG. This may have significant advantages regarding dosing,etc. Further, a more “natural”; or more “human” product may be moredesirable to the patient, who may desire therapy, although in a senseartificial, to be as “natural” as possible. There may be otheradvantages (e.g., pharmacokinetic advantages) in a recombinant hCGproduct having carbohydrate (e.g., glycan) structure which is closer tonatural (e.g., human urinary) hCG than other recombinant products.

The invention is thus a recombinant version of hCG which carries a mixof α2,3 and α2,6 sialic acid and therefore more closely resemblesnatural hCG. It is expected that the use of this compound for controlledovarian stimulation, in IVF techniques, and ovulation induction willresult in a more natural stimulation of the ovary compared to existingrecombinant products.

According to the present invention there is provided recombinant hCG(“rhCG” or “rechCG”) (and/or a recombinant hCG preparation) includingα2,3 sialylation and α2,6 sialylation. The rhCH or rhCG preparation mayoptionally further include α2,8 sialylation.

Herein term “recombinant hCG preparation” includes a preparation for,e.g., pharmaceutical use which includes recombinant hCG. In embodimentsof the invention, the rhCG may be present as a single isoform or as amixture of isoforms.

The rhCG (or rhCG preparation) according to the invention may have asialic acid content [expressed in terms of a ratio of moles of sialicacid to moles of protein] of 15 mol/mol or greater (Example 8), forexample of from 15 mol/mol to 25 mol/mol, for example from 17 mol/mol to24 mol/mol, for example from 17.7 mol/mol to 23 mol/mol, for examplefrom 18 mol/mol to 22 mol/mol, for example from 19 mol/mol to 21mol/mol, for example from 19 mol/mol to 20 mol/mol. The rhCG of theinvention may be produced or expressed in a human cell line.

The rhCG (or rhCG preparation) according to the invention may have 10%or more of the total sialylation being α2,3-sialylation. For example,20, 30, 40, 45, 50, 55, 60, 70, 80 or 90% or more of the totalsialylation may be α2,3-sialylation. The rhCG (or rhCG preparation) mayinclude α2,3-sialylation in an amount which is from 45% to 80% of thetotal sialylation, for example 50% to 70% of the total sialylation, forexample 55 to 65% of the total sialylation. The rhCG (or rhCGpreparation) may include α2,3-sialylation in an amount which is from 65to 85% of the total sialylation, for example from 70 to 80% of the totalsialylation, for example from 71 to 79% of the total sialylation. TherhCG (or rhCG preparation) of the invention may have 50% or less of thetotal sialylation being α2,6-sialylation. For example 45, 40, 30, 20,10, 5% or less of the total sialylation may be α2,6-sialylation. TherhCG (or rhCG preparation) may include α2,6-sialylation in an amountwhich is from 20-55% of the total sialylation, for example, 30-50% ofthe total sialylation, for example 35-45% of the total sialylation. TherhCG (or rhCG preparation) may include α2,6-sialylation in an amountwhich is from 15 to 35% of the total sialylation, for example from 20 to30% of the total sialylation, for example from 21 to 29% of the totalsialylation. The rhCG (or rhCG preparation) of the invention may have 5%or less of the total sialylation being α2,8-sialylation. For example2.5% or less of the total sialylation may be α2,8-sialylation. The rhCG(or rhCG preparation) may include α2,8-sialylation in an amount which isfrom 0 to 4% of the total sialylation, for example 0.1 to 4% of thetotal sialylation, for example from 0.5 to 3% of the total sialylation,for example from 0.5 to 2.5% of the total sialylation. The rhCG (or rhCGpreparation) of the invention may have no α2,8-sialylation. Bysialylation it is meant the amount of sialic residues present on the hCGcarbohydrate structures. α2,3-sialylation means sialylation at the 2,3position (as is well known in the art) and α2,6 sialylation at the 2,6position (also well known in the art). Thus “% of the total sialylationmay be a 2,3 sialylation” refers to the % of the total number of sialicacid residues present in the hCG which are sialylated in the 2,3position. The term “% of the total sialylation being α2,6-sialylation”refers to the % of the total number of sialic acid residues present inthe hCG which are sialylated in the 2,6 position.

The rhCG (or rhCG preparation) according to the invention may have asialic acid content (amount of sialylation per hCG molecule) of (basedon the mass of protein, rather than the mass of protein pluscarbohydrate) of 6% or greater (e.g., between 6% and 15%, e.g., between7% and 13%, e.g., between 8% and 12%. e.g., between 11% and 15%, e.g.,between 12% and 14%) by mass.

Recombinant hCG expressed in Chinese hamster ovary (CHO) cells includesexclusively a 2, 3 sialylation.

The rhCG of the invention may be produced or expressed in a human cellline. This may simplify (and render more efficient) the productionmethod because manipulation and control of, e.g., the cell growth mediumto retain sialylation may be less critical than with known processes.The method may also be more efficient because there is less basic rhCGproduced than in production of known rhCG products; more acidic rhCG isproduced and separation/removal of basic hCG is less problematic. TherhCG may be produced or expressed in a PER.C6® cell line, a PER.C6®derived cell line or a modified PER.C6® cell line. The cell line may bemodified using α2,3-sialyltransferase. The rhCG may include α2,6-linkedsialic acids (α2,6 sialylation) provided by endogenous sialyltransferase activity {of the cell line]. Alternatively or additionally,the cell line may be modified using α2,6-sialyltransferase.

The rhCG may be produced using α2,3-sialyltransferase. The rhCG mayinclude α2,6-linked sialic acids (α2,6 sialylation) provided byendogenous sialyl transferase activity. The rhCG may be produced usingα2,3- and/or α2,6-sialyltransferase.

According to the present invention in a further aspect there is provideda method of production of rhCG and/or an rhCG preparation as describedherein (according to aspects of the invention) comprising the step ofproducing or expressing the rhCG in a human cell line, for example aPER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6®cell line, for example a cell line which has been modified usingα2,3-sialyltransferase.

The rhCG structure contains glycan moieties. Branching can occur withthe result that the glycan may have 1, 2, 3, 4 or more terminal sugarresidues or “antennae”, as is well known in the art. The rhCG of theinvention may have glycans with sialylation presence on mono-antennaryand/or di-antennary and/or tri-antennary and/or tetra-antennarystructures. The rhCG may include mono-sialylated, di-sialylated,tri-sialylated and tetra-sialylated glycan structures, for example withrelative amounts as follows: 0.1-4% mono-sialylated; 35-45%di-sialylated; 0.5-8% tri-sialylated and 0-1% tetra-sialylated (e.g., asshown by WAX analysis of charged glycans, as set out in Example 8 D).Preferably, the recombinant hCG, of the invention includes mono (1S),di(2S), tri(3S) and tetra(4S) sialylated structures. Preferably, therelative amounts of sialylated structures are in the following ratios(1S:2S:4S:4S): 0.2-1%: 35-40%: 2.5-7%: 0.5-1% (e.g., as shown by WAXanalysis of charged glycans, as set out in Example 8 D).

According to the present invention in a further aspect there is providedrhCG produced (e.g., expressed) in a human cell line. The rhCG mayinclude α2,3- and α2,6-sialylation. The rhCG may be produced orexpressed in a PER.C6® cell line, a PER.C6® derived cell line or amodified PER.C6® cell line. The cell line may be modified usingα2,3-sialyltransferase. The rhCG may include α2,6-linked sialic acids(α2,6 sialylation) provided by endogenous sialyl transferase activity[of the cell line]. Alternatively or additionally, the cell line may bemodified using α2,6-sialyltransferase. The rhCG (or rhCG preparation)according to the invention may have a sialic acid content [expressed interms of a ratio of moles of sialic acid to moles of protein] of 15mol/mol or greater, for example of from 15 mol/mol to 25 mol/mol, forexample of from 17 mol/mol to 24 mol/mol, for example from 17.7 mol/molto 23 mol/mol, for example from 18 mol/mol to 22 mol/mol, for examplefrom 19 mol/mol to 21 mol/mol, for example from 19 mol/mol to 20mol/mol. The rhCG (or rhCG preparation) may have 10% or more of thetotal sialylation being α2,3-sialylation, for example 45% to 80% of thetotal sialylation may be α2,3-sialylation, for example 50% to 70% of thetotal sialylation may be α2,3-sialylation, for example 55 to 65% of thetotal sialylation may be α2.3-sialylation. For example 65-85% of thetotal sialylation may be α2,3-sialylation. The rhCG (or rhCGpreparation) of the invention may have 50% or less of the totalsialylation being α2,6-sialylation. For example, 20-55% of the totalsialylation may be α2,6-sialylation, for example, 30-50% of the totalsialylation may be α2,6-sialylation, for example, 35-45% of the totalsialylation may be α2,6-sialylation. For example 15-35% of the totalsialylation may be α2,6-sialylation. The rhCG (or rhCG preparation) ofthe invention may have 5% or less of the total sialylation beingα2,8-sialylation, for example 0 to 4%, e.g., 0.5-4% of the totalsialylation may be α2,8-sialylation. The rhCG (or rhCG preparation) ofthe invention may have no α2,8-sialylation.

According to the present invention in a further aspect there is provideda pharmaceutical composition comprising rhCG including α2,3-sialylationand α2,6-sialylation (e.g., as set out above). The pharmaceuticalcomposition may further comprise FSH and/or LH.

FSH can be obtained by any means known in the art. FSH, as used herein,includes human-derived and recombinant FSH. Human-derived FSH can bepurified from any appropriate source (e.g., urine) by any method knownin the art. The FSH may be recombinant FSH for example expressed in ahuman cell line. Methods of expressing and purifying recombinant FSH arewell known in the art.

LH can be obtained by any means known in the art. LH, as used herein,includes human-derived and recombinant LH. Human-derived LH can bepurified from any appropriate source (e.g., urine) by any method knownin the art. Methods of expressing and purifying recombinant LH are knownin the art.

The pharmaceutical composition may be for the treatment of infertility,e.g., for use in, e.g., assisted reproductive technologies (ART),ovulation induction or intrauterine insemination (IUI). Thepharmaceutical composition may be used, for example, in medicalindications where known hCG preparations are used. The present inventionalso provides the use of rhCG and/or an rhCG preparation describedherein (according to aspects of the invention) for, or in themanufacture of a medicament for, the treatment of infertility. Thepharmaceutical compositions of the present invention can be formulatedinto well-known compositions for any route of drug administration, e.g.,oral, rectal, parenteral, transdermal (e.g., patch technology),intravenous, intramuscular, subcutaneous, intrasusternal, intravaginal,intraperitoneal, local (powders, ointments or drops) or as a buccal ornasal spray. A typical composition comprises a pharmaceuticallyacceptable carrier, such as aqueous solution, non-toxic excipients,including salts and preservatives, buffers and the like, as described inRemington's Pharmaceutical Sciences fifteenth edition (Matt PublishingCompany, 1975), at pages 1405 to 1412 and 1461-87, and the nationalformulary XIV fourteenth edition (American Pharmaceutical Association,1975), among others.

Examples of suitable aqueous and non-aqueous pharmaceutical carriers,diluents, solvents or vehicles include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol. and the like),carboxymethylcellulose and suitable mixtures thereof, vegetable oils(such as olive oil), and injectible organic esters such as ethyl,oleate.

The compositions of the present invention also can contain additivessuch as but not limited to preservatives, wetting agents, emulsifyingagents, and dispersing agents. Antibacterial and antifungal agents canbe included to prevent growth of microbes and includes, for example,paraben, chlorobutanol, phenol, sorbic acid, and the like. Furthermore,it may be desirable to include isotonic agents such as sugars, sodiumchloride, and the like.

In some cases, to effect prolonged action it is desirable to slow theabsorption of hCG (and other active ingredients, if present) fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of hCG then depends uponits rate of dissolution which, in turn, can depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered hCG combination form is accomplished by dissolving orsuspending the hCG combination in an oil vehicle.

Injectable depot forms can be made by forming microencapsule matrices ofthe hCG (and other agents, if present) in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of hCG to polymerand the nature of the particular polymer employed, the rate of hCGrelease can be controlled. Examples of other biodegradable polymersinclude polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc.Depot injectable formulations are also prepared by entrapping the hCG inliposomes or microemulsions which are compatible with body tissues.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium justprior to use. Injectable formulations can be supplied in any suitablecontainer, e.g., vial, pre-filled syringe, injection cartridges, and thelike.

Injectable formulations can be supplied as a product havingpharmaceutical compositions containing hCG (optionally with FSH, LHetc.) if there is more than one active ingredient (i.e., hCG and e.g.,FSH or LH) these may be suitable for administration separately ortogether. if administered separately, administration can be sequential.The product can be supplied in any appropriate package. For example, aproduct can contain a number of pre-filled syringes containing eitherhCG, FSH, or a combination of both FSH and hCG, the syringes packaged ina blister package or other means to maintain sterility. A product canoptionally contain instructions for using the hCG and FSH formulations.

The pH and exact concentration of the various components of thepharmaceutical composition are adjusted in accordance with routinepractice in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICALBASIS FOR THERAPEUTICS, 7^(th) ed. In a preferred embodiment, thecompositions of the invention are supplied as compositions forparenteral administration. General methods for the preparation of theparenteral formulations are known in the art and are described inREMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY. supra, at pages780-820. The parenteral compositions can be supplied in liquidformulation or as a solid which will be mixed with a sterile injectablemedium just prior to administration. In an especially preferredembodiment, the parenteral compositions are supplied in dosage unit formfor ease of administration and uniformity of dosage.

The present invention will now be described in more detail withreference to the following Examples and to the attached drawings.

Sequence Selection 1. Human hCG

The coding region of the gene for the hCG alpha polypeptide was usedaccording to Fiddes and Goodman (1979). The sequence is banked asAH007338 and at the time of construction there were no other variants ofthis, protein sequence. The nucleic acid sequence is referred to hereinas SEQ ID 1 and the polypeptide sequence is referred to as SEQ ID NO:5.

The coding region of the gene for hCG beta polypeptide was usedaccording to Fiddes and Goodman (1980). The sequence is banked asNP_000728 and is consistent with the protein sequences of CGbeta3,CGbeta5 and CGbeta7. The nucleic acid sequence is referred to herein asSEQ ID 2 and the polypeptide sequence is referred to as SEQ ID NO:6.

2. Sialyltransferase

α2,3-Sialyltransferase—The coding region of the gene forbeta-galactoside alpha-2,3-sialyltransferase 4 (α2,3-sialyltransferase,ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequenceis banked as L23767. The nucleic acid sequence is referred to herein asSEQ ID NO:3 and the polypeptide sequence is referred to as SEQ ID NO:7.

α2,6-Sialyltransferase—The coding region of the gene forbeta-galactosamide alpha-2,6-sialyltransferase 1(α2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al.(1990). The sequence is banked as NM_003032. The nucleic acid sequenceis and referred to herein as SEQ ID NO: 4 and the polypeptide sequenceis referred to as SEQ ID NO:8.

EXAMPLES Example 1 Construction of the hCG Expression Vector

The coding sequence of hCG alpha polypeptide (AH007338, SEQ ID 1) andhCG beta polypeptide (NP_000728, SEQ ID NO:2) were amplified by PCRusing the primer combinations CGa-fw and CGa-rev and CGb-fw and CGb-recrespectively.

CGa-fw (SEQ ID NO: 9) 5′-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3′;CGa-rev (SEQ ID NO: 10) 5′-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3′; CGb-fw(SEQ ID NO: 11) 5′-CCAGGCGCGCCACCATGGAGATGTTCCAGGGGCTGC-3′; and CGb-rev(SEQ ID NO: 12) 5′-CCGGGTTAACTTATTGTGGGAGGATCGGGG-3′;

The resulting amplified hCG beta DNA was digested with the restrictionenzymes AscI and HpaI and inserted into the Awl and HpaI sites on theCMV driven mammalian expression vector carrying a neomycin selectionmarker. Similarly the hCG alpha DNA was digested with BamHI and NheI andinserted into the sites BamHI and NheI on the expression vector alreadycontaining the hCG beta polypeptide DNA.

The vector DNA was used to transform the DH5α strain of E. coli.Colonies were picked for amplification and, of the number which includedthe vector containing both hCG alpha and beta, twenty were selected forsequencing. All colonies selected for sequencing contained the correctsequences according to SEQ ID NO:1 and SEQ ID NO:2. Plasmid phCG A+B wasselected for transfection (FIG. 1).

Example 2 Construction of the ST3 Expression Vector

The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4(ST3, L23767, SEQ ID NO:3) was amplified by PCR using the primercombination 2,3STfw and 2,3STrev.

2,3STfw (SEQ ID NO: 13) 5′-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3′; and2,3STrev (SEQ ID NO: 14) 5′-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3′.

The resulting amplified ST3 DNA was digested with the restrictionenzymes BamHI and AflII and inserted into the BamHI and AflII sites onthe CMV driven mammalian expression vector carrying a hygromycinresistance marker. The vector was amplified as previously described andsequenced. Clone pST3#1 (FIG. 2) contained the correct sequenceaccording to SEQ ID NO:3 and was selected for transfection.

Example 3 Construction of the ST6 Expression Vector

The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1(ST6, NM_003032, SEQ ID NO:4) was amplified by PCR using the primercombination 2,6STfw and 2,6STrev.

2,6STfw (SEQ ID NO: 15) 5′-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3′; and2,6STrev (SEQ ID NO: 16) 5′-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3′.

The resulting amplified ST6 DNA was digested with the restrictionenzymes BamHI and AflII and inserted into the BamHI and AflII sites onthe CMV driven mammalian expression vector carrying a hygromycinresistance marker. The vector was amplified as previously described andsequenced. Clone pST6#11 (FIG. 3) contained the correct sequenceaccording to SEQ ID NO:4 and was selected for transfection.

Example 4 Stable Expression of phCG A+B in PER.C6® Cells, TransfectionIsolation and Screening of Clones

PER.C6 clones producing hCG were generated by expressing bothpolypeptide chains of hCG from a single plasmid (see Example 1).

To obtain stable clones a liposome based transfection agent was usedwith the phCG A-1-B construct. Stable clones were selected in PER.C6®selection media supplemented with 10% FCS and containing G418. Threeweeks after transfection G418 resistant clones grew out. A total of 389clones were selected for isolation. The isolated clones were cultured inselection medium until 70-80% confluent. Supernatants were assayed forhCG protein content using an hCG selective ELISA and pharmacologicalactivity at the hCG receptor in cloned cell line, using a cAMPaccumulation assay. Clones (118) expressing functional protein wereprogressed for culture expansion to 24 well, 6 well and T80 flasks.

Studies to determine productivity and quality of the material from 47clones were initiated in T80 flasks to generate sufficient material.Cells were cultured in supplemented media as previously described for 7days and the supernatant harvested. Productivity was determined usingthe hCG selective ELISA. The isoelectric profile of the material wasdetermined (using the method described in Example 6). The informationfrom the IEF was used to select clones for metabolic clearance rateanalysis. Clones with sufficient productivity and quality were selectedfor sialyltransferase engineering.

Example 5a Level of Sialylation is Increased in Cells that Over Expressα2,3-Sialyltransferase. Stable Expression of pST3 in hCG ExpressingPER.C6® Cells; Transfection Isolation and Screening of Clones

PER.C6 clones producing highly sialylated hCG were generated byexpressing α2,3 sialyltransferase from separate plasmids (see Example 2)in PER.C6® cells already expressing both polypeptide chains of hCG (seeExample 4). Clones produced from PER.C6® cells as set out in Example 4were selected for their characteristics including productivity, goodgrowth profile, production of functional protein, and produced hCG whichincluded some sialylation.

Stable clones were generated as previously described in Example 4.Clones from the α2,3-sialyltransferase program were isolated, expandedand assayed. The final clone number for the α2,3-study was five. Theα2,3-sialyltransferase clones were adapted to serum free media andsuspension conditions.

As before, clones were assayed using a hCG selective ELISA, functionalresponse in an hCG receptor cell line, IEF (Example 6). They were alsoassessed for metabolic clearance rate (Example 9) and USP hCG Bioassay(Example 10). Results were compared to a commercially availablerecombinant hCG (OVITRELLE®, Serono) and the parental hCG PER.C6® celllines. Representative samples are shown in the Examples and Figures.

In conclusion expression of hCG together with α2,3-sialyltransferase inPER.C6® cells results in increased levels of sialylated hCG compared tocells expressing hCG only.

Example 5b Stable Expression of pST3 in hCG Expressing PER.C6® Cells—aDifferent Method

The alpha beta heterodimer produced above (Example 4) had a low level ofsialylation resulting in a very basic IEF profile. As indicated above(Example 5a) expression of hCG together with α2,3-sialyltransferase inPER.C6® cells results in increased levels of sialylated hCG compared tocells expressing hCG only.

A double transfection of the hCG alpha and beta subunit genes togetherwith the α2,3 sialyltransferase enzyme gene into PER.C6® cells insuspension cell culture format was performed. Cell lines were generatedby co-transfecting the hCG vector (dual alpha/beta, Example 1) and thevector encoding α2,3-sialyltransferase (Example 2) under serum freeconditions. Clones produced from PER.C6® cells were selected for theircharacteristics including productivity, good growth profile, productionof functional protein, and produced hCG which included some sialylation.Clones were isolated, expanded and assayed.

As before, clones were assayed using an hCG selective ELISA, functionalresponse in an hCG receptor cell line, IEF (Example 6). They were alsoassessed for metabolic clearance rate (Example 9) and USP hCG Bioassay(Example 10). Results were compared to a commercially availablerecombinant hCG (OVITRELLE®, Serono) and the parental hCG PER.C6® celllines. Representative samples are shown in the Examples and Figures (seeExamples 6, 9, 10, FIGS. 4 and 5). The recombinant hCG produced by theclones (that is, recombinant hCG according to the invention) hassignificantly improved sialylation (i.e., on average more hCG isoformswith high numbers of sialic acids), compared to hCG expressed withoutα2,3-sialyltransferase and OVITRELLE® (see Examples 6 and 8, FIG. 4).

Example 6 Analysis of the Isoelectric Point pI of PER.C6® Cell ProducedhCG Isoforms by Isoelectric Focussing

Electrophoresis is defined as the transport of charged molecules througha solvent by an electrical field. The mobility of a biological moleculethrough an electric field will depend on the field strength, net chargeon the molecule, size and shape of the molecule, ionic strength andproperties of the medium through which the molecules migrate.

Isoelectric focusing (IEF) is an electrophoretic technique for theseparation of proteins based on their pI. The pI is the pH at which aprotein has no net charge and will not migrate in an electric field. Thesialic acid content of the hCG isoforms subtly alters the pI point foreach isoform, which can be exploited using this technique to visualisethe PER.C6® cell produced hCG isoforms from each done.

The isoelectric points of the PER.C6® cell produced hCG isoforms in cellculture supernatants were analyzed using isoelectric focussing. Cellculture media from PER.C6 cell produced hCG clones were produced asdescribed in Example 4, 5a and 5b.

PER.C6® cell produced hCG samples were separated on NOVEX® IEF Gelscontaining 5% polyacrylamide under native conditions on a pH 3.0-7.0gradient in an ampholyte solution pH 3.0-7.0. Proteins were visualisedusing Coomassie Blue staining, using methods well known in the art.

FIG. 4 shows the detection of rhCG Isoforms by IEF stained withCoomassie Blue in compositions according to the invention (Track 3, 10μg, and Track 4, 15 μg) and the CHO derived composition of the priorart, OVITRELLE® (Track 1, OVITRELLE®, 10 μg, and Track 2, OVITRELLE®, 15μg). The bands represent isoforms of hCG containing different numbers ofsialic acid molecules. Using this method clones producing hCG isoformswith a higher number of sialic acid molecules were identified. FIG. 4indicates that human cell line derived recombinant hCGs engineered withα2,3-sialyltransferase (compositions according to the invention) have amore acidic profile than OVITRELLE®.

Example 7 Analysis of the Sialic Acid Linkages of PER.C6® Cell ProducedhCG

Glycoconjugates were analyzed using a lectin based glycandifferentiation method. With this method glycoproteins andglycoconjugates bound to nitrocellulose can be characterized. Lectinsselectively recognize a particular moiety, for example α2,3 linkedsialic acid. The lectins applied are conjugated with the steroid haptendigoxigenin which enables immunological detection of the bound lectins.

Purified PER.C6® cell produced from a parental clone (no additionalsialyltransferase), and a α2,3-sialyltransferase engineered clone wereseparated using standard SDS-PAGE techniques. A commercially availablerecombinant hCG (OVITRELLE®, Serono) was used as a standard.

Sialic acid was analyzed using the DIG Glycan Differentiation Kit (Cat.No. 11 210 238 001, Roche) according to the manufacturers instructions.Positive reactions with Sambucus nigra agglutinin (SNA) indicatedterminally linked (2-6) sialic acid. Positive reactions with Maackiaamurensis agglutinin II (MAA): indicated terminally linked (α2-3) sialicacid

In summary the parental clone contained low levels of both α2,3- andα2,6-sialic acid. The clones engineered with α2,3-sialyltransferasecontained high levels of α2,3-sialic acid linkages and low levels ofα2,6-sialic acid linkages. The standard control OVITRELLE® only containsα2,3-sialic acid linkages. This is consistent with what is known aboutrecombinant proteins produced in Chinese Hamster ovary (CHO) cells(Kagawa et al., 1988, Takeuchi et al., 1988, Svensson et al., 1990).

In conclusion, engineering of PER.C6® hCG cells withα2,3-sialyltransferase successfully increased the number of sialic acidmolecules conjugated to the recombinant hCG in the sample.

Examples 8A and 8B Quantification of Total Sialic Acid

Sialic acid is a protein-bound carbohydrate considered to be amono-saccharide and occurs in combination with other mono-saccharideslike galactose, mannose, glucosamine, galactosamine and fucose. Thetotal sialic acid on purified rhCG according to the invention wasmeasured using a method based on the method of Stanton et. al. (J.Biochem. Biophys. Methods. 30 (1995), 37-48).

Example 8A

The total sialic acid content of PER.C6® cell produced recombinant hCGmodified with α2,3-sialyltransferase (e.g., Example 5a, Example 5b) wasmeasured and found to be greater than 15 mol/mol, [expressed in terms ofa ratio of moles of sialic acid to moles of protein], for examplegreater than 18 mol/mol, for example 19.1 mol/mol. This can be comparedto OVITRELLE®, which has total sialic acid content of 17.6 mol/mol.

Example 8B

The total sialic acid content of PER.C6® cell produced recombinant hCGmodified with α2,3-sialyltransferase 080019-19 (prepared by the methodsof Example 5b above) was measured and found to be 20 mol/mol, [expressedin terms of a ratio of moles of sialic acid to moles of protein]. Again,this may be favourably compared with OVITRELLE®, which has total sialicacid content of 17.6 mol/mol. This Example (080019-19) was tested toquantify the relative amounts of α2,3 and α2,6 sialic acid (Example 8C).

Example 8C—Quantification of Relative Amounts of α2,3 and α2,6 SialicAcid

The relative percentage amounts of α2,3 and α2,6 sialic acid on purifiedrhCG [Example (080019-19), and two other Examples prepared by themethods of Example 5] were measured using known techniques—HPLC withNormal-phase (NP).

To quantify the alpha 2,3 and 2,6 sialic acid in 0-link glycans thefollowing analysis was performed. The O-linked glycans were cleaved fromthe hCG sample using an Orela Glycan Release Kit and separated onNP-HPLC. Samples of the extracted, pooled, glycans (extracted as above)were digested with different sialidases to determine the linkages. ThisEnzymatic degradation of glycans was performed using alpha 2-3,6,8sialidase and alpha 2-3, sialidase. The enzymatic digested glycans werethen re-separated on the NP column, and the O-Glycans were identified onthe NP-HPLC using prepared standards. The relative percentages werecalculated and are shown in the following table (SA=Sialic Acid).

% SA Structure 080019-19 09PD-84-04 09PD84-006-3 α 2,3 SA 59 63 63 α 2,6SA 41 37 37

The relative percentages were found to be in the ranges 55%-65% (e.g.,59%) for α2,3 sialylation; and 35 to 45% (e.g., 41%) for α2,6sialylation.

Example 8D Quantification of Relative Amounts Mono, Di, Tri and TetraAntennary Sialylated Structures

The relative percentage amounts of mono, di, tri and tetra sialylatedstructures on glycans extracted from purified rhCG (the three samplesused in Example 8C) were measured using known techniques.

Each sample of rhCG was immobilized (gel block), washed, reduced,alkylated and digested with PNGase F overnight. The N-glycans were thenextracted and processed. N-glycans for NP-HPLC and WAX-HPLC analysiswere labelled with the fluorophore 2AB as detailed in Royle et al.

Weak anion exchange (WAX) HPLC to separate the N-glycans by charge(Example 8C) was carried out as set out in Royle et al., with a FetuinN-glycan standard as reference. Glycans were eluted according to thenumber of sialic acids they contained. All samples included mono (1S),di(2S), tri(3S) and tetra(4S) sialylated structures. The relativeamounts of sialylated structures were found to be in the followingratios (1S:2S:4S:4S): 0.1-4%:35-45%:0.5-8%:0-1%.

A preferred example, 080019-19, included mono (15), di(2S), tri(3S) andtetra(4S) sialylated structures. The relative amounts of sialylatedstructures were in the following ratios (15:25:45:45):0.1-4%:35-45%:0.5-8%:0-1%.

Example 9 Determination of the Metabolic Clearance Rates of rhCG

To determine the metabolic clearance rate (MCR) of PER.C6® cell producedhCG samples engineered using α2,3-sialyltransferase (e.g., Example 5a,5b), conscious female rats (3 animals per clone) were injected into thetail vein at time zero with a bolus of rhCG (1-10 μg/rat, based on ELISAquantification of samples, DRG EIA 1288). Blood samples (400 μl) weretaken from the tip of the tail at 1, 2, 4, 8, 12, 24 and 32 hours aftertest sample injection. Serum was collected by centrifugation and assayedfor hCG content by ELISA (DRG EIA 1288). The MCR of PER.C6® cellproduced hCG samples engineered using α2,3-sialyltransferase showed thatthe half-life was similar to the standard (FIG. 5). FIG. 6 shows thatother hCG samples engineered using α2,3-sialyltransferase may haveimproved half-life compared to the standard (FIG. 6).

Example 10—hCG Bioassay According to USP

A hCG Bioassay was carried out, to assay the hCG specific activity. Theactivity was measured according to USP (USP Monographs: ChorionicGonadotropin, USPC Official Aug. 1, 2009-11/30/09), using OVITRELLE® asa standard. OVITRELLE® has a biological activity of 26,000 IU/mg (Curr.Med. Res. Opin. 2005 December; 21(12): 1969-76). The acceptance limitwas >21,000 IU hCG/mg. The biological activity for a sample of humancell line derived hCG recombinant hCG engineered withα2,3-sialyltransferase (having sialic acid content 19.1 mol/mol—seeExample 8) was 27,477 IU hCG/mg.

Example 11 Production and Purification Overview

A procedure was developed to produce recombinant hCG in PER.C6® cellsthat were cultured in suspension in serum free medium. The procedure isdescribed below and was applied to several hCG-producing PER.C6® celllines.

Recombinant hCG from an α2,3-clone was prepared using a using amodification of the method described by Lowry et al. (1976).

For the production of PER.C6® cell-produced hCG, the cell lines wereadapted to a serum-free medium, i.e., EX-CELL® 525 (JRH Biosciences).The cells were first cultured to form a 70%-90% confluent monolayer in aT80 culture flask. On passage the cells were re-suspended in the serumfree medium, EX-CELL′ 525+4 mM L-Glutamine, to a cell density of 0.3×10⁶cells/mi. A 25 ml cell suspension was put in a 250 ml shaker flask andshaken at 100 rpm at 37° C. at 5% CO₂. After reaching a cell density of>1×10⁶ cells/ml, the cells were sub-cultured to a cell density of 0.2 or0.3×10⁶ cells/ml and further cultured in shaker flasks at 37° C. 5% CO₂and 100 rpm.

For the production of hCG, the cells were transferred to a serum-freeproduction medium, i.e., VPRO Biosciences), which supports the growth ofPER.C6® cells to very high cell densities (usually >10⁷ cells/ml in abatch culture). The cells were first cultured to >1×10⁶ cells/nil inEX-CELL® 525, then spun down for 5 min at 1000 rpm and subsequentlysuspended in VPRO medium+6 mM L-glutamine to a density of 1×10⁶cells/mi. The cells were then cultured in a shaker flask for 7-10 daysat 37° C., 5% CO₂ and 100 rpm. During this period, the cells grew to adensity of >10⁷ cells/mi. The culture medium was harvested after thecell viability started to decline. The cells were spun down for 5 min at1000 rpm and the supernatant was used for the quantification andpurification of hCG. The concentration of hCG was determined using ELISA(DRG EIA 1288).

Thereafter, purification of hCG was carried out using a modification ofthe method described by Lowry et al. (1976). This was achieved bychromatography on DEAE cellulose, gel filtration on SEPHADEX® G100,adsorption chromatography on hydroxyapatite, and preparativepolyacrylamide electrophoresis.

During all chromatographic procedures, the presence of immunoreactiverecombinant hCG was confirmed by RIA (DRG EIA 1288) and IEF (Example 6).

FIGURE LEGENDS

FIGS. 1, 2 and 3: Plasmid maps of the phCGalpha/beta, pST3 and pST6expression vectors. CMV=Cytomegalovirus promoter, BGHp(A)=Bovine GrowthHormone poly-adenylation sequence, fl ori=fl origin of replication,SV40=Simian Virus 40 promoter, Neo=Neomycin resistance marker,Hyg=Hygromycin resistance marker, SV40 p(A)=Simian Virus 40poly-adenylation sequence, hCG α=human chorionic gonadotropin alphapolypeptide, hCG β=human chorionic gonadotropin beta polypeptide,ST3GAL4=α2,3-sialyltransferase, ST6GAL1=α2,6-sialyltransferase,ColEI=ColEI origin of replication, Amp=ampicillin resistance marker.

FIG. 4. Detection of rhCG Isoforms by IEF stained with Coomassie Blue incompositions according to the invention (Track 3, 10 μg, and Track 4, 15μg) and the CHO derived composition of the prior art. OVITRELLE® (Track1, OVITRELLE®, 10 μg, and Track 2, OVITRELLE®, 15 μg). The bandsrepresent isoforms of hCG containing different numbers of sialic acidmolecules. FIG. 4 indicates that human cell line derived recombinanthCGs engineered with α2,3-sialyltransferase (compositions according tothe invention) have a more acidic profile than OVITRELLE®.

FIG. 5. Metabolic clearance rates of rhCG samples produced byα2,3-sialyltransferase engineered PER.C6 cells. Samples were chosen fortheir sialic acid content based on their IEF profile. Female rats (3animals per clone) were injected into the tail vein at time zero with abolus of rhCG (1-10 peat). Blood samples collected over time wereassayed for hCG content by ELISA.

FIG. 6 Long term Metabolic clearance rates of rhCG samples produced byα2,3-sialyltransferase engineered PER.C6® cells. Female rats (3 animalsper clone) were injected into the tail vein at time zero with a bolus ofrhCG (1-10 peat). Blood samples collected over time were assayed for hCGcontent by ELISA.

REFERENCES

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Human Chorionic Gonadotropin Alpha Polypeptide

Accession number AH007338

Nucleotide sequence of hCG alpha (SEQ ID NO: 1)   1ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT  61GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG 301GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA AProtein sequence of hCG alpha (SEQ ID NO: 5)   1MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP  61ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS 121YCSFGEMKE

Human Chorionic Gonadotrophin Beta Polypeptide

Accession number NP_000728

Nucleotide sequence of hCG beta (SEQ ID NO: 2)   1ATGGAGATGT TCCAGGGGCT GCTGCTGTTG CTGCTGCTGA GCATGGGCGG GACATGGGCA  61TCCAAGGAGC CGCTTCGGCC ACGGTGCCGC CCCATCAATG CCACCCTGGC TGTGGAGAAG 121GAGGGCTGCC CCGTGTGCAT CACCGTCAAC ACCACCATCT GTGCCGGCTA CTGCCCCACC 181ATGACCCGCG TGCTGCAGGG GGTCCTGCCG GCCCTGCCTC AGGTGGTGTG CAACTACCGC 241GATGTGCGCT TCGAGTCCAT CCGGCTCCCT GGCTGCCCGC GCGGCGTGAA CCCCGTGGTC 301TCCTACGCCG TGGCTCTCAG CTGTCAATGT GCACTCTGCC GCCGCAGCAC CACTGACTGC 361GGGGGTCCCA AGGACCACCC CTTGACCTGT GATGACCCCC GCTTCCAGGA CTCCTCTTCC 421TCAAAGGCCC CTCCCCCCAG CCTTCCAAGT CCATCCCGAC TCCCGGGGCC CTCGGACACC 481CCGATCCTCC CACAATAA Protein sequence of hCG beta (SEQ ID NO: 6)   1MEMFQGLLLL LLLSMGGTWA SKEPLRPRCR PINATLAVEK EGCPVCITVN TTICAGYCPT  61MTRVLQGVLP ALPQVVCNYR DVRFESIRLP GCPRGVNPVV SYAVALSCQC ALCRRSTTDC 121GGPKDHPLTC DDPRFQDSSS SKAPPPSLPS PSRLPGPSDT PILPQ

Beta-Galactoside Alpha-2,3-Sialyltransferase 4

Accession Number L23767

Nucleotide sequence of ST3GAL4 (SEQ ID NO: 3)   1ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG  61TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG 121AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 181TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 241TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA 301GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 361GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 421GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 481AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 541AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 601ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC 661TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT 721GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG 781GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 841GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 901AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 961CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of ST3GAL4(SEQ ID NO: 7)   1MCPAGWKLLA MLALVLVVMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN  61YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV 121VVGNGHRLRN SSLGDAINKY DVVIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN 181NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA 241DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL 301KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF

Beta-Galactosamide Alpha-2,6-Sialyltransferase 1

Accession number NM_003032

Nucleotide sequence of ST6GAL1 (SEQ ID NO: 4)    1ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT   61GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG  121CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC  181CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT  241CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC  301TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC  361AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG  421CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC  481AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT  541TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA  601GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA  661CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG  721AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT  781GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC  841TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG  901CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC  961CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT 1021TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC 1081GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG 1141CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC 1201TTCCGGACCA TTCACTGCTA A Protein Sequence of ST6GAL1 (SEQ ID NO: 8)   1MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS  61QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN 121KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP 181WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 241KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 301PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 361DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC

1. A pharmaceutical composition comprising recombinant human chorionicgonadotropin (hCG), wherein the recombinant hCG in the compositioncomprises α2,3- and α2,6-sialylation, and wherein the pharmaceuticalcomposition further comprises FSH.
 2. A pharmaceutical compositionaccording to claim 1, wherein the recombinant hCG in the composition isglycosylated and comprises mono-(1S), di-(2S), tri-(3S) and tetra-(4S)sialylated glycan structures.
 3. A pharmaceutical composition accordingto claim 1, wherein the recombinant hCG is produced or expressed in ahuman cell line.
 4. A pharmaceutical composition according to claim 3,wherein the cell line is modified using α2,3-sialyltransferase.
 5. Apharmaceutical composition according to claim 3, wherein the recombinanthCG includes α2,6 sialylation provided by endogenous sialyl transferaseactivity.
 6. A pharmaceutical composition according to claim 3, whereinthe cell line is a PER.C6 cell line deposited with the EuropeanCollection of Cell Cultures under accession number
 96022940. 7. Apharmaceutical composition according to claim 2, wherein the recombinanthCG has relative amounts of sialylated structures in the ratio 0.2-1mono-sialylated structures: 35-40 di-sialylated structures: 2.5-7tri-sialylated structures: 0.5-1 tetra-hsialylated structures.
 8. Amethod of production of a pharmaceutical composition comprisingrecombinant hCG (rhCG) including α2,3-sialylation and α2,6-sialylation,comprising expressing hCG in a human cell line.
 9. A method according toclaim 8, wherein the cell line has been modified with α2,3-sialyltransferase.
 10. A method according to claim 8, wherein the recombinanthCG includes α2,6-sialylation provided by endogenous sialyl transferaseactivity.
 11. A method according to claim 8, wherein the cell line is aPER.C6 cell line deposited with the European Collection of Cell Culturesunder accession number 96022940.